L-threonate ferrous, as well as pharmaceutical composition and use for improving and treating human anemia thereof

ABSTRACT

A compound of ferrous L-threonate with structure (I), its compostions and methods useful for iron supplementation for mammals, particularly for human body to improve and treat nutritional iron-deficiency anemia, blood loss anemia and hemolytic anemia.

This application is a 371 of PCT/CN98/00174, filed on Aug. 28, 1998.

TECHNICAL FIELDS OF INVENTION

This invention relates to a new derivative of L-threonic acid as apharmaceutical active compound. More specifically, the inventionconcerns ferrous L-threonate, a method of making the same, apharmaceutical composition thereof. In a further aspect, this inventionrelates to use of ferrous L-threonate for the preparation of apharmaceutical composition for improving and treating the anemiadiseases, particularly, nutritional iron-deficiency anemia (IDA)(hypoferric anemia), blood-loss anemia (hemorrhagic anemia) andhemolytic anemia.

TECHNICAL BACKGROUNDS OF INVENTION

L-threonic acid is one of metabolites of vitamin C. It is reported thatsome physiological functions of vitamin C were exerted very possibly bysome of its metabolites, for example, L-threonic acid and the like. Inthe other hand, the existing of these compounds could profoundlyinfluence the uptake and utilization of vitamin C.

Calcium L-threonate is a derivative of L-threonic acid. CalciumL-threonate can improve uptake of vitamin C by the lymphoma cells andcan be used as the high efficient calcium nutrient for preventing andtreating varied diseases caused by calcium deficiency (Chinese Patent,ZL 96 06507.9). Also, calcium L-threonate is useful for allayinginflammation and reducing blood pressure. But there was no report forother salts of L-threonic acid and their use as drugs on treatingdiseases.

Anemia is a common disease, which is caused by a various reasons. Theirreasons mainly include: (1) a decrease of red blood corpuscle (RBC) involume or in quantity; (2) a decrease of hemoprotein in amount due tochronic or acute blood loss and the injury of RBC due to its exposing tosome chemical compounds; and (3) a reduction of the number of producedRBC due to deficiency of marrow or such nutrients as ferrous and vitaminB₁₂ used to produce RBC.

Nutritional iron-deficiency anemia (IDA) is a very common case amonganemia diseases. The reason is that the body iron balance betweenexcretion and storage has not been sustained. Consequently, there wasmore and more excretion but less and less storage of body iron withtime. These phenomena often happened during the periods of growth,pregnancy and chronic blood loss due to various reasons. For example theless intake of iron from food is an important factor to lead to irondeficiency for body.

In fact, the nutritional IDA is a worldwide nutritional problem. Fortreating this kind of disease, simply taking iron-rich foods is hard tohave good effects compared with using drugs. Among IDA drugs of ironpreparations, ferrous sulfate is a relative popular drug with goodeffectiveness, low price and abundant resource. Even so, the extensiveapplication of ferrous sulfate for treating IDA is limited because ofits side effects (Chinese Child Blood, Vol. 1, p24-26, 1996). Therefore,it is desirable to provide new active compounds and compositions thereoffor improving and treating IDA very efficiently.

Blood-loss anemia is a usual case of anemia. Most patients are women inthe periods of pregnancy and menses.

Another anemia is hemolytic anemia. It is mainly caused by the followingreasons: exposing to hemolytic chemical materials such as naphthaleneand sulfanilamide; production of antibody in cells due to administrationof drugs; and presence of cells with hereditary defection in body. Amethod for treating hemolytic anemia is to remove those harmful chemicalreagents from body. Therefore, it is desirable to provide an activecompounds and compositions thereof so as to improve and treat hemolyticanemia. It is in particularly desirable to provide an active compoundand composition thereof so as to improve and cure these three kinds ofanemia diseases.

OBJECT OF INVENTION

It is one object of this invention to provide a new derivative ofL-threonic acid, in particularly, ferrous L-threonate.

It is another object of the invention is to provide compositionscontaining ferrous L-threonate for improving and treating such anemia asnutritional IDA, blood-loss anemia and hemolytic anemia.

It is a further object of the invention is to provide a method forimproving and treating these anemia diseases.

ADVANTAGE OF INVENTION

Ferrous L-threonate of this invention has higher absorption ratecompared with known iron preparations, for example, ferrous sulfate.Under the same dosages (element Fe/kg body weight) and the same bodysituation, animal test results showed that ferrous L-threonate wasabsorbed with higher absorptivity than ferrous sulfate. It is usedsafely and without toxicity. As iron preparation, ferrous L-threonatecan significantly improve and cure hemolytic anemia, blood-loss anemiaand nutritional IDA with the characters of high bioavailability andcontrolled release, compared with ferrous gluconate and ferrousfumarate. Ferrous L-threonate can be taken as both good iron supplementand drug for improving and treating anemia disease.

DETAIL DESCRIPTION OF INVENTION

This invention concerns the compound so-called ferrous L-threonate withchemical structure given as below.

Ferrous L-threonate has L-form optical structure. It is stable, solublein water and exists mainly as the complex form in its solution.

Preparation methods for ferrous L-threonate:

1. Prepared by neutralization reaction of L-threonic acid with ferrousoxides or ferrous hydroxides, for example, with ferrous oxide (FeO) andferrous hydroxide (Fe(OH)₂);

2. Prepared by replacement reaction of L-threonic acid or calciumL-threonate with inorganic ferrous salts, such as ferrous sulfate(FeSO₄), ferrous chloride (FeCl₂) and ferrous nitrate (Fe(NO₃)₂),wherein L-threonic acid can be obtained by oxidizing vitamin C or byremoving calcium from calcium L-threonate.

Ferrous oxides, ferrous hydroxides and inorganic ferrous salts can beprepared from the corresponding ferric compounds, such as, ferricnitrate, ferric sulfate and Fe3O4. If L-threonic acid is available inthe market, it also can be used to prepare ferrous L-threonate accordingto the methods described above.

The reaction conditions for preparing ferrous L-threonate are,

(1) Under pH 6-10, preferably at pH 7-9, vitamin C was oxidized byoxidant, for example, by hydrogen peroxide solution to get a solution ofL-threonic acid,

(2) Under the protection of inert gas such as nitrogen gas, the solutionof L-threonic acid reacts with ferrous compounds, such as ferrousoxides, ferrous hydroxides and ferrous salts to produce ferrousL-threonate.

This invention also relates to compositions containing ferrousL-threonate as therapeutically active component. The composition alsocan include other therapeutically active compounds, pharmaceuticallyacceptable carrier and such medical supplementary materials as flavours,excipients and vitamins. Preferably, the composition of the inventioncontains ferrous L-threonate, vitamin C or vitamin B12, of which theweight ratio of ferrous L-threonate is 10-90%, preferred range is 30-80%and more preferred range is 40-60%.

The definition of the term “pharmaceutically acceptable” is that fromthe view of the point of pharmacy, those materials or components andtheir properties are acceptable by patients and pharmacists withstability and bioavailability.

The ferrous L-threonate of the invention can be prepared as any forms ofdrug. These forms include tablet, capsule, suppository, liquid,suspending agent, syrup, emulsion, gel, ointment, freeze-dried powder,pill, film, lipoplast, dispersible micro-powder and injection solution.Solid forms of tablet and powder are preferred.

According to the invention, the tablet can be prepared by mixing theactive compound, ferrous L-threonate with excipients like calciumcarbonate, calcium phosphate and lactose, with disintegrating agent likecorn starch, with cohesive materials like starch and gelatin, withlubricants like magnesium stearate and talc powder, and with somecontrolled release materials like carboxypolymethylene and carboxymethylcellulose.

Inner tablets used for preparing sugarcoating tablets can be made in thesimilar methods described above. Sugarcoating materials are insect glue,arabic gelatin, talc titanium dioxide and so on. Usually, several-layertechnique was employed to prepare the inner tablets to avoid theincompatible properties possibly caused by different components used.

Injection solution can be prepared according to the common technology.Similarly, other forms of drug for ferrous L-threonate's composition canbe prepared in terms of the known techniques and methods.

Ferrous L-threonate can be taken as nutritional additive to food and tobeverage to enhance the uptake quantity of iron by body and thus toimprove and cure anemia disease.

The invention provides methods for improving and treating such anemia asnutritional IDA, blood-loss anemia and hemolytic anemia by ferrousL-threonate and/or its compositions.

In a further respect, the methods of the invention need patients to takea certain quantity of ferrous L-threonate.

According to methods of the invention, methods are available to animal,such as bull, horse and sheep and especially to human being includingchild, woman and old people, and particularly to child and woman.

Different ways of administration, such as oral, injecting and so on, donot influence the activity and effectiveness of ferrous L-threonate.

According to methods of the invention, the dosages for treating anemiawill vary in terms of patient conditions and administration ways used inpractice. To determine the accurate dosage needs to consider theanemia's extent, patient's age, body weight and so on. For instance, thedosage for children should be less than that for adult.

On using solid form of drug, the quantity of ferrous L-threonate foradult is usually in the range of 10 mg-1 g a day. Preferred range is 20mg-800 mg a day, more preferred range 30 mg-500 mg a day, for example,30 mg, 250 mg or 400 mg, far more preferred range 50 mg-200 mg a day,for example, 60 mg or 100 mg or 150 mg.

For animal, particularly for mammal except human being, the dosage offerrous L-threonate depends on the experimental results for thedifferent races of animals.

The methods of the invention for improving and treating anemia areusable for nutritional IDA, blood-loss anemia and hemolytic anemia.

More specifically, the methods are suitable to growth period forchildren, pregnancy and menses periods for women, the case of chronic oracute blood loss and the case of insufficient uptake of iron from food.

The methods of the invention also can be used to the case of hemolyticanemia developed by RBC damage and decrease of RBC number when RBCexposes to some chemical reagents, for example.

This invention also relates to the functions of ferrous L-threonate assupplementary drug under some conditions, to treat the whole body systemdisorder caused by, for instance, iron deficiency.

In terms of evaluation program of food safety and toxicology, Hornmethod cited in the published standard of GB15193.4-94 was adopted toevaluate the acute toxicity of ferrous L-threonate with the results ofLD₅₀ 3.16 g/kg and 3.69 g/kg for female and male rat, respectively. Thisfact illustrated that the toxicity of ferrous L-threonate belongs to thegrade of very lower.

According to the published standards of GB15193.4-94, GB15193.5-94,GB15198.4-94 and GB15193.14-94, the Ames test, micronucleus test of bonemarrow cells and chromosome aberration test of mice testicle cells havebeen carried out and all tests' results are negative. Oraladministration of ferrous L-threonate did not produce embryonic toxicityand abberation.

The invention supplies the digestive absorption study of ferrousL-threonate on male Wistar rat (see experiment 1).

This invention also describes the pharmacodynamics studies of ferrousL-threonate for hemolytic anemia model, nutritional IDA model andblood-loss anemia model on rats (see experiment 2).

This invention further gives the clinical investigation of ferrousL-threonate for improving IDA disease of children (see experiment 3).

EXPERIMENT 1 Metabolic and Absorption Study of Ferrous L-threonate onMale Wistar Rats

Drugs: Ferrous L-threonate was supplied by Beijing Juneng Asia-PacificLife Science Research Center. It is yellow-green powder and soluble inwater and contains element ferrous 15.47%. Its chemical formula isFe(C₄H₇O₅)₂.2H₂O, molecular weight:362.

Animals: Male Wistar rats were supplied by Experimental Animal Center,Tumor Institute of Heilongjian Province with body weight of 140 g-180 g.The iron-low diet contains iron 0.9 mg/kg.

Methods

50 rats were randomly divided into 5 groups of [19] 10 rats each. Onthis basis of administration of iron-low diet, each group of rats wasadministered respectively by gavage according to the dosage below:

High dosage group: Fe, 5 mg/kg, ferrous L-threonate, 32.32 mg/kg;

Middle dosage group: Fe, 2.5 mg/kg, ferrous L-threonate, 16.16 mg/kg;

Low dosage group: Fe, 1.0 mg/kg, ferrous L-threonate, 6.46 mg/kg;

Positive drug control group (ferrous sulfate): Fe, 2.5 mg/kg, ferroussulfate, 12.43 mg/kg;

Negative drug control group (iron-low diet): Fe, 0.9 mg/kg (measuredvalue).

After the end of 7 days feeding normal diet period, rats weresupplemented with iron-low diet prepared according to AOAC formula foranother 7 days. The 1-3 groups were treatment groups and supplementedwith ferrous L-threonate 32.32 mg/kg (5 mgFe/kg, high dose group), 16.16mg/kg (2.5 mg Fe/kg, middle dose group) and 6.46 mg/kg (1.0 mg Fe/kg,low dose group), respectively. The forth, positive drug control group,was provided with ferrous sulfate 12.43 mg/kg (2.5 mg Fe/kg). The fifthwas model control group and was offered with iron-low diet (0.9 mgFe/kg). Each groups were arranged to take drug one time by I.G (gavage)method per day and in the same time were supplemented with deionizedwater.

The test was sustained three days. Each rat's excrement was collected,dried at temperature 80-90° C., weighed and ground into 40-60 meshes.About 0.5 to 0.6 gram sample was taken and a mixture of nitric acid andperchloric acid at a ratio of 4:1 was added therein until completion ofdigestion. The constant volume is 10 ml. The iron content of excrementwas determined by atomic absorption method and the iron absorptive ratefor each rat can be calculated by the formula listed below.${{Digestive}\quad {absorptivity}\quad (\%)} = {\frac{\begin{matrix}{{{Quantity}\quad {of}\quad {iron}\quad {intake}} -} \\{{Quantity}\quad {of}\quad {iron}\quad {excrement}}\end{matrix}}{{Quantity}\quad {of}\quad {iron}\quad {intake}} \times 100}$

Results: The tests results were listed in Table 1 and it indicated thatthe digestive absorptivity of ferrous L-threonate is significantlyhigher compared with that of ferrous sulfate (P<0.01) in the conditionof same ferrous dosage.

TABLE 1 Digestive absorptivity for different ferrous salts Dosage GroupsSamples (mg Fe/kg) Absorptivity (%) High dosage of 10 5 42.60 ± 1.79ferrous L-threonate Middle dosage of 10 2.5  54.90 ± 4.08* ferrousL-threonate Low dosage of 10 1 59.10 ± 5.01 ferrous L-threonate Ferroussulfate 10 2.5 42.00 ± 1.02 group Model group 10 0.9 55.50 ± 1.85

EXPERIMENT 2 Pharmacodynatics of Ferrous L-threonate for HemolyticAnemia Model, Nutritional IDA Model and Blood-loss Anemia Model

1. Drugs: Ferrous L-threonate, yellow-green powder and 15.47% ferrouscontent, was supplied by Beijing Juneng Asia-Pacific Life ScienceResearch Center. Ferrous gluconate, the positive control drug,yellow-green powder and 11% ferrous content, was purchased from GanjianPharmaceutical Company of Jianxi Province. Ferrous fumarate, 6.4%ferrous content, was purchased from the 18^(th) Pharmaceutical Companyof Shanghai. All drugs were diluted by distilled water.

2. Testing animal and division of group Male Wistar rats (Animal Centerof Beijing Medical University) with body weight 250±10 g were kept undercontrolled conditions and supplemented with normal diet and water.Animal diet also was purchased from Beijing Medical University.

(1) Hemolytic Anemia Model: Hemolytic anemia model was prepared for ratsby injecting reagent of acetylphenylhydrazine (C₈H₁₀N₂O, produced byBeijing Biochemical Company) subcutaneously. The drugacetyphenylhydrazine was mixed with physiological saline to get itsinjection solution of 4% by weight of acetyphenylhydrazine. The solutionwas injected subcutaneously into each rat with a dosage of 57 μg/g atthe beginning of the experiments. The content of blood hemochrome foreach rat was examined at 2 days and 4 days later, respectively. When themean blood hemochrome decreased to about 6.11±0.25 g %, rats wererandomly divided into 6 groups of 18 rats each. The 1-3 groups weretreatment groups of ferrous L-threonate with high, middle and lowdosages. The forth groups was positive drug control groups. The fifthgroup and the sixth group were designed as model group and normalcontrol group, respectively.

(2) Nutritional Anemia Model: Male Wistar rats with body weight 43±2.5 g(Animal Center of Beijing Medical University) were randomly divided into8 groups of 10 rats each, wherein 6 groups were established for theinvestigation of nutritional anemia and 2 groups were used as normalcontrol group. The rats of these six groups were supplemented withiron-low diet (prepared according to the formula of AOAC, iron content8.0 mg/kg) and deionized water four weeks to prepare the nutritional IDAmodel, which also was called hemoglobin exhausting test. Another twogroups were designed as normal control groups and were provided withnormal diet (obtained from Beijing Medical University) and water duringthe whole experimental period.

Hemoglobin of each group of rats was measured one time every 7 days.After the end of 4 weeks of feeding iron-low diet period, the content ofhemoglobin of nutritional IDA groups' rats reduced to about 7.51±0.17 gand in that time the blood index of one nutritional IDA group and onenormal control group were determined as index of before treatment bydrug. Other 6 groups (three for ferrous L-threonate, one for ferrousgluconate, one for mode control and one normal control group) rats werekept under controlled conditions (ambient temperature 23° C., 60%relative humidity, and 10 hours light) in animal feeding room tocontinue the further test.

(3) Blood-loss Anemia Model: 120 male Wistar rats (Animal Center ofBeijing Medical University) with body weight 130±8.2 g were randomlydivided into 8 groups of 15 rats each. 7 groups were prepared ashemolytic anemia groups and one group was taken as normal control groupsupplemented with normal diet and water. The hemolytic anemia model wasprepared by subjecting rats to inner canthus bloodletting of right eyethrough capillary glass tube. The first bloodletting content was 2.0 mlfor each rat and in that time the range of hemochrome for rats was from9.78 to 10.65 g %. Two days later, the second bloodletting (2 ml/rat)was carried out with the hemochrome values of 7.40-7.88 g %. The totalbloodletting volume for each rat was 4 ml and was 32.9% in volume of thewhole blood circulating the body.

While one hemolytic anemia group and one normal control group were takenas reference groups compared with other groups left. These two groups'rats were killed and the blood parameters were recorded. Other sixgroups now were randomly divided into three treatment groups of ferrousL-threonate with high, middle and low dosages respectively, two positivedrugs control groups of ferrous gluconate and ferrous fumarate and onemodel group. At the end of 12 days and 18 days experimental periods, 5and 10 rats of each group were killed correspondingly. During the courseof experiment, the variations of hemochrome content were examined with 6days interval.

Dosages: Three treatment groups of ferrous L-threonate were divided intohigh dosage group with element ferrous 17.28 mg/kg, middle dosage groupwith element ferrous 8.64 mg/kg and low dosage group with elementferrous 4.32 mg/kg. While Element ferrous content 8.64 mg/kg as ferrousgluconate or ferrous fumarate was for positive drug control group. Thegroups for blood-loss anemia were provided iron-low diet and deionizedwater during the period of the administration of drugs, while the normalanimal control group was provided normal diet and deionized water.

Test Results: The index of anemia were investigated for the cases ofbefore anemia model preparation, after anemia model preparation, one,two or three weeks after treatment of drugs.

(1) Results for Hemolytic Model Study

TABLE 2 Variations of hemoglobin content of groups (g %) after two weeksof administration of drugs Two weeks Before After One week after Groupsanemia anemia after treatment treatment High dosage of 14.9 ± 6.18 ±11.95 ± 14.34 ± ferrous L- 0.422 0.419 0.844 0.420 threonate Middledosage of 15.01 ± 5.87 ± 11.11 ± 14.04 ± ferrous L- 0.421 0.590 0.6330.383 threonate Low dosage of 14.89 ± 5.94 ± 9.95 ± 12.57 ± ferrous L-0.501 0.526 0.488 0.590 threonate Ferrous gluconate 15.34 ± 6.57 ± 10.62± 12.65 ± group 1.176 0.272 0.591 0.530 Model group 15.03 ± 6.01 ± 8.14± 10.79 ± 0.352 0.503 0.530 0.364 Normal control 14.60 ± 14.23 ± 14.33 ±14.21 ± group 0.302 0.434 0.526 0.354

TABLE 3 Results of RBC counts (× 10¹²/L) Before Two weeks Groups anemiaAfter anemia after treatment High dosage of ferrous 7.185 ± 0.292 2.310± 0.248 5.663 ± 0.236 L-threonate Middle dosage of 7.238 ± 0.181 2.163 ±0.422 5.376 ± 0.273 ferrous L-threonate Low dosage of ferrous 8.290 ±0.188 3.370 ± 0.193 4.863 ± 0.136 L-threonate Ferrous gluconate 7.215 ±0.212 2.188 ± 0.201 5.011 ± 0.337 group Model group 8.180 ± 0.277 3.323± 0.384 3.647 ± 0.293 Normal control group 7.160 ± 0.251 7.132 ± 0.3247.202 ± 0.336

TABLE 4 Results of blood granulophilocyte counts (%) Two weeks Cells forBefore after Groups counting anemia After anemia treatment High dosageof ferrous 1000 1.193 ± 94.80 ± 6.11 ± L-threonate 0.093 1.183 1.375Middle dosage of 1000 1.138 ± 95.35 ± 7.60 ± ferrous L-threonate 0.1982.026 0.551 Low dosage of ferrous 1000 1.298 ± 95.475 ± 9.35 ±L-threonate 0.147 1.575 1.089 Ferrous gluconate 1000 1.448 ± 96.00 ±9.12 ± group 0.139 2.174 0.966 Model group 1000 1.135 ± 97.85 ± 19.21 ±0.256 1.790 3.063 Normal control group 1000 1.602 ± 1.734 ± 1.932 ±0.154 0.253 0.387

The results listed in table 2-4 showed that by treating two weeks, theitems of RBC and hemochrome for all iron supplementation groupsincreased significantly compared with those of before treatment, theblood granulophilocyte counts lowered than treatment before, while thecases of high and middle dosages of ferrous L-threonate were similar tothose of normal control group. In contrast to drugs groups, model groupbehaved relative weak effects on curing anemia and this in fact wasreasonable. Under the same dosage of iron (8.64 mg/kg), the actions offerrous L-threonate on treating anemia was considerably better thanferrous gluconate.

TABLE 5 Contents of whole blood iron and serum ferritin (SF) Ironcontent of whole blood Serum ferrintin Groups (μg/ml) ng/ml Beforeanemia 361.89 ± 20.841 23.60 ± 1.94 After anemia 178.81 ± 14.849  9.52 ±1.61 High dosage of 326.95 ± 26.956 24.05 ± 1.74 Ferrous L-threonateMiddle dosage of 301.89 ± 19.500 23.54 ± 2.50 ferrous L-threonate Lowdosage of 283.53 ± 23.051 19.38 ± 1.57 Ferrous L-threonate Ferrousgluconate 286.04 ± 26.797 20.14 ± 2.83 Model group 198.11 ± 17.122 13.70± 0.95 Normal control group 333.11 ± 17.35  23.48 ± 1.82

The results from table 5 showed that in comparison with model group, thecontents of whole blood iron and serum ferritin for all groups of drugstreatment enhanced significantly (P<0.01) while variations of theseitems for ferrous L-threonate groups except low dosage group were moreconsiderably better than those of ferrous gluconate group.

(2) Results for Nutritional IDA Model Study

The exhaustive hemoglobin test of rat was 28 days period and the resultswere listed in table 6 and table 7.

TABLE 6 Hemoglobin exhausting test for rats supplemented with iron-lowdiet n = 10 (g %) Groups 7-day 14-day 21-day 28-day High dosage 11.19 ±10.28 ± 9.03 ± 7.82 ± group with low-iron 0.41 0.48 0.75 0.37 dietMiddle dosage 11.33 ± 10.14 ± 9.06 ± 7.51 ± group with low-iron 0.380.57 0.83 0.57 diet Low dosage 11.18 ± 10.82 ± 8.45 ± 7.36 ± group withlow 0.41 0.35 0.53 0.58 iron diet known drug with 10.87 ± 10.44 ± 8.64 ±7.34 ± low iron diet 0.45 0.48 0.39 0.46 control group 11.35 ± 10.08 ±8.74 ± 7.53 ± with low-iron diet 0.60 0.69 0.73 0.60 control group 11.60± 12.30 ± 12.85 ± 13.76 ± with normal diet 0.46 0.42 0.57 0.30

TABLE 7 Results of RBC, blood granulophilocyte (GPC) counts, blood ironcontent and SF of hemoglobin exhausting test RBC Blood iron Groups (×10¹²/L) GPC (%) (μg/ml) SF (ng/ml) Normal diet 6.654 ± 0.46  3.41 ± 0.32313.11 ± 7.35  23.48 ± 1.82 group The sixth  2.82 ± 0.41 11.96 ± 0.88168.63 ± 23.21 12.99 ± 1.61 iron-low diet group

TABLE 8 Variations of hemochrome content for IDA rats after treatment n= 10 g % Groups 7-day 14-dat 21-day Normal control group 13.70 ± 0.32**13.84 ± 0.22** 13.98 ± 0.39** Model group  7.92 ± 0.61  7.77 ± 0.44 8.31 ± 0.51 High dosage of ferrous 10.52 ± 0.49* 12.23 ± 0.45** 13.67 ±0.41** L-threonate Middle dosage of 10.12 ± 0.66* 11.46 ± 0.36* 12.98 ±0.38** ferrous L-threonate Low dosage of ferrous  8.74 ± 0.44 10.76 ±0.37* 11.87 ± 0.50** L-threonate Ferrous gluconate  9.08 ± 0.65 11.66 ±0.54* 12.40 ± 0.58** group *P < 0.05 **P < 0.01 compared with modelgroup

TABLE 9 RBC, GPC, SF and blood iron for IDA rats after treatment 21 daysn = 10 RBC Blood iron SF Groups (× 10¹²/L) GPC (%) (μg/ml) (ng/ml)Normal 7.197 ± 0.28  2.30 ± 0.35 308.54 ± 17.38  24.2 ± 1.06 controlgroup Model group 3.273 ± 0.23 12.30 ± 0.87 200.31 ± 28.78  13.1 ± 1.45High dosage 6.564 ± 0.65  2.29 ± 0.54 304.37 ± 14.12 23.40 ± 1.50 offerrous L-threonate Middle 6.116 ± 0.21  2.63 ± 0.76 302.02 ± 18.9220.88 ± 1.53 dosage of ferrous L-threonate Low dosage 5.957 ± 0.44  4.18± 1.12 290.78 ± 20.13 17.98 ± 1.34 of ferrous L-threonate Ferrous 6.286± 0.36  2.36 ± 0.79 297.50 ± 14.82 20.39 ± 2.10 gluconate group

The results of table 8 showed that the hemochrome content of IDA ratstreated by ferrous L-threonate started to increase at the end of 7 dayssupplementation, and reached nearly the normal levels of normal controlgroups.

RBC, whole blood iron content and SF for drug treatment groups (seetable 9) increased considerably, while the blood granulophilocyte countsof these groups decreased compared with the model control group.Further, under the same ferrous dosage the situations of parametersinvestigated were better than those of the known drug, ferrousgluconate, which suggested that ferrous L-threonate has better effectson improving and treating nutritional IDA.

(3) Results for Blood-loss Model Study

TABLE 10 Blood hemochrome content for rats with blood-loss anemia n = 106 days 12 days 18 days Before after after after Groups treatmenttreatment treatment treatment High dosage of 7.88 ± 0.28 8.70 ± 0.4311.01 ± 0.34 12.40 ± 0.63 ferrous L-threonate Middle dosage 7.40 ± 0.548.35 ± 0.21 10.67 ± 0.37 12.14 ± 0.27 of ferrous L-threonate Low dosageof 7.61 ± 0.53 8.00 ± 0.42 10.20 ± 0.49 11.26 ± 0.79 ferrous L-threonateFerrous 7.50 ± 0.76 7.95 ± 0.30 10.50 ± 0.61 11.80 ± 0.63 gluconategroup Ferrous 7.43 ± 0.85 7.80 ± 0.24 10.12 ± 0.52 11.84 ± 0.45 fumarategroup Model group 7.51 ± 0.63 7.42 ± 0.26  9.03 ± 0.56 10.39 ± 0.46

TABLE 11 Variations of RBC and GPC after treatment n = 10 RBC (× 10¹²/L)GPC (%) Groups 12 days 18 days 12 days 18 days High dosage of 5.67 ±5.84 ± 10.04 ± 7.84 ± ferrous L-threonate 0.313 0.199 0.97 0.871 Middledosage of 4.810 ± 5.21 ± 12.58 ± 8.44 ± ferrous L-threonate 0.178 0.1941.54 0.753 Low dosage of 4.242 ± 4.76 ± 13.12 ± 8.91 ± ferrousL-threonate 0.128 0.228 1.84 1.277 Ferrous gluconate 4.488 ± 5.14 ±12.66 ± 9.04 ± group 0.271 0.342 2.04 0.793 Ferrous fumarate 4.200 ±5.14 ± 12.66 ± 9.04 ± group 0.271 0.342 2.04 0.793 Model group 4.046 ±4.37 ± 16.50 ± 11.58 ± 0.147 0.310 1.47 1.10

TABLE 12 Variations of whole blood iron content and SF after treatment n= 10 Whole blood iron (μg/ml) SF (ng/ml) Groups 12 days 18 days 12 days18 days High 5.567 ± 0.490 8.973 ± 0.952 13.81 ± 0.3  24.12 ± 0.77dosage of ferrous L- threonate Middle 5.304 ± 0.594 8.879 ± 0.890 12.36± 0.21 22.91 ± 0.68 dosage of ferrous L- threonate Low 4.508 ± 0.2356.384 ± 1.188 10.90 ± 0.75 18.70 ± 1.16 dosage of ferrous L- threonateFerrous 5.450 ± 0.376 7.027 ± 0.799 12.12 ± 0.11 22.33 ± 1.33 gluconategroup Ferrous 4.583 ± 0.794 6.446 ± 0.745 11.46 ± 0.71 19.67 ± 0.98fumarate group Model 3.358 ± 0.286 5.077 ± 0.937  8.13 ± 0.44 13.87 ±1.41 group

Results listed in table 10-12 showed that under the same ferrous dosage,ferrous L-threonate has better effects on treating IDA compared withboth ferrous gluconate and ferrous fumarate.

EXPERIMENT 3 The Clinical Study of Ferrous L-threonate on Improving theCases of IDA for Children

Drugs: Ferrous L-threonate tablets, white color and 7.5 mg ferrouscontent and 30 mg vitamin C each tablet, was supplied by Beijing JunengAsia-Pacific Life Science Research Center.

Subjects: Thirty school-age children aged from 8 to 13 years of Haerbincity of China.

Methods: Blood samples of 10 μl from ear and 5 ml from arm vein for eachpatient were collected for measurement.

(1) Measurement of RBC: 10 μl blood sample was added to 2 ml diluent andthe mixture was shaken completely. One drop of this suspending solutiontaken by circle glass bar was poured into counting cell. RBC wascountered through microscope by laying counting plate on the microstat.

(2) Measurement of HB: The ferric cyanide method was used to determinethe HB. 10 μl ear blood sample was added to 5 ml acid diluent to obtainthe completely mixed solution by shaking. HB was measured by SHhemoglobinometer manufactured by Huauang Instrument Company of JianshuProvince. Ferric cyanide was purchased from Medical ExaminationInstitute of Shanghai.

(3) Measurement of FEP: To add 0.05 ml whole blood sample anticoagulatedby heparin to 3.5 ml of acid anhydrous ethyl alcohol, the mixture wasshaken for 5 min and then was centrifugalized with speed of 3000 r/min.The centrifugate of mixture was used for the measurement of fluorometricmethod (400 nm for exciting optical filter and 600 nm for emittingoptical filter).

According to formula given below, the FEP can be calculated.${{FEP}\left( {{{µg}/L}\quad {RBC}} \right)} = {35 \times \frac{Fu}{PCV}}$

Where Fu and PCV represent fluorescence extent and hematocrit,respectively.

(4) Measurement of SF: SF reagent box was purchased from TianjingJiuding Biotechnology company and SN-682 radio-immune γ-ray counter wasmanufactured by Rihuan Instrument Company, Nuclear Medical Institute ofShanghai.

To centifugalized 2 ml whole blood sample, the upper solution was usedfor measurement of SF through the method of radio-immunecompetition-inhibition program.

Test steps:

(1) Determination of Anemia: Based on the standards of WHO and Chinainvestigation of prevention and treatment for IDA, Hb<120 g/L, FEP>500μg/L, SF<16 μg/L and RBC<4 million/mm³, together with the clinicalexamination, 39 males and 22 females were diagnosed as IDA patients(Hb<120 g/L) from 300 boys and 274 girls.

Among these 62 IDA patients, 30 children were randomly selected astreatment group of ferrous L-threonate.

Another 30 children with IDA were selected from another prime schoolwith the same diagnosing standards and were established as controlgroup.

(2) Intervention test: Child with IDA did usually not like activity,concentrated their attention difficultly and showed apathetic, sleepy,poor appetite and pale skin.

Observation time for treatment group and control group was 30 daysperiod and during that period, there were no any intervention onchildren's activity and food.

(3) Dosages: Each child of treatment group was administered ferrousL-threonate tablets twice a day, one tablet each time. While normalcontrol group was treated with placebo by the same method.

Results:

TABLE 13 Variations of Hb and FEP before and after treatment Beforetreatment ({overscore (x)} ± S) After treatment ({overscore (x)} ± S)Groups Hb (g/L) Hb (g/L) FEP (μg/L) FEP (μg/L) Treatment 104.8 ± 6.1 669.1 ± 72.0 120.4 ± 11.2 445.1 ± 80.9 group Control group 102.8 ± 6.7 754.5 ± 76.4 103.6 ± 9.1  748.9 ± 85.5

TABLE 14 Variations of SF and RBC before and after treatment Beforetreatment ({overscore (x)} ± S) After treatment ({overscore (x)} ± S)RBC RBC Groups (μg/L) (10⁴/mm³) SF (μg/L) (10⁴/mm³) Treatment 12.45 ±1.50 387 ± 6.67 17.01 ± 1.99 401.5 ± 12.4 group Control group 12.0 ± 0.9377 ± 9.7  12.7 ± 1.5  380 ± 8.8

The results of Hb, SF and RBC listed in table 13 and table 14 measuredbefore and after treatment showed that the variations of each item fortreatment group reaches the level of understandingly significantdifference (P<0.01). Also, after 30 days of treatment by ferrousL-threonate, these variations of treatment group are significantlybetter compared with those of control group (P<0.01).

TABLE 15 Effectiveness of supplementation of ferrous L-threonate Hb >120 g/L No and ΔHb > 5 g/L effectiveness Groups ΔHb > 10 g/L (%) (%) (%)Total (%) Treatment 17 7 6 30 group (56.7%) (23.3%) (20.0%)  (100.0%)Control  0 0 30   0 group (0%) (0%) (100.0%) (100.0%)

Thus, the effective rate of improving the case of IDA for children byferrous L-threonate is as high as 80%.

EXAMPLE 1 Preparation of Ferrous L-threonate

0.1 mol vitamin c was solved in 500 ml water distilled twice. Thesolution was adjusted to weak alkaline (pH=7-9) by 0.1 mol/L sodiumhydroxide (NaOH) and the hydrogen peroxide (H₂O₂) of concentration 30%was added to it drop by drop in the condition of stirring. The mixturewas kept for 20 min at about 10° C., and then treated with activatedcarbon, and heated at 80° C. to decompose the excess of hydrogenperoxide. When evolution of oxygen had ceased, the hot mixture wasfiltered, and the filtrate was cooled to room temperature for furtherprocessing. Under the protection of inert gas, for example, nitrogen gas(N₂), 0.15 mol ferrous sulfate (FeSO₄.2H₂O) was added to filtrate in asmall portion gently. The reaction mixture was stirred two hours, thefiltered and the filtrate was concentrated under diminished pressure.The pale green crystals that formed in the course of several hours wereseparated by filtration and finally were recrystallized twice fromdistilled water. The product obtained in this way was ferrousL-threonate with two crystal water molecules, C₈H₁₄O₁₀Fe.2H₂O.

Anal. Calc. For C₈H₁₄O₁₀Fe.2H₂O: C 26.52, H 5.01, Fe 15.42. Found: C26.66, H 4.91, Fe 15.23.

When treating Fe(C₄H₇O₅)₂.2H₂O at 10 mmHg diminished pressure and 50° C.temperature, the ferrous L-theronate with one crystal water molecule wasobtained.

Anal. Calc. For C₈H₁₄O₁₀Fe.H₂O: C 27.91, H 4.69, Fe 16.23. Found: C27.66, H 4.94, Fe 16.53.

Anhydrous ferrous L-threonate was prepared by drying Fe(C₄H₇O₅)₂.H₂O at5 mmHg diminished pressure and 120° C. temperature.

Anal. Calc. For C₈H₁₄O₁₀Fe: C 29.45, H 4.33, Fe 17.13. Found: C 29.11, H4.39, Fe 17.60.

EXAMPLE 2 Composition of Ferrous L-threonate Tablet

Composition for 1000 tablets: Ferrous L-threonate 48.5 g Vitamin C 30 gMannitol 180 g Starch 80 g Magnesium stearate right amount Perfumerycompound right amount

The components above were granlulated, dried and molded to obtain 1000tablets of Ferrous L-threonate.

What is claimed is:
 1. A compound having the formula of

or hydrate thereof.
 2. A pharmaceutical composition comprising apharmaceutically effective amount of a compound having the formula

or a hydrate thereof and a pharmaceutically acceptable carrier.
 3. Thepharmaceutically composition according to claim 2, wherein said compoundis present in an amount of about 10-90% by weight of the composition. 4.The pharmaceutical composition according to claim 1, further comprisingvitamin C and/or vitamin B¹².
 5. A method for treating anemia in amammal, comprising administering to the mammal a pharmaceuticallyeffective amount of a ferrous L-threonate compound having the formula

or a hydrate thereof.
 6. The method according to claim 5, wherein theferrous L-threonate compound is administered at a dosage of from about0.01 to 1 gram per day.
 7. The method of claim 5, wherein the anemia isnutritional iron-deficiency (hypoferric) anemia, blood-loss(hemorrhagic) anemia, or hemolytic anemia.
 8. The method of claim 5,wherein the mammal is a human.
 9. A method for supplying iron to amammal, comprising administering to the mammal a pharmaceuticallyeffective amount of a compound having the formula

or a hydrate thereof.
 10. A method for preventing anemia in a mammal,comprising administering to the mammal a pharmaceutically effectiveamount of a compound having the formula

or a hydrate thereof.
 11. A process for the preparation of a compoundhaving the formula

or a hydrate thereof, comprising reacting L-threonate acid with aferrous compound selected from the group consisting of inorganic ferroussalts, ferrous oxides and ferrous hydroxides.
 12. A process forpreparing a compound having the formula

or a hydrate thereof, comprising: reacting calcium L-threonate with aninorganic salt of ferrous under conditions so as to provide ferrousL-threonate.